Full-thickness tissue engineered oral mucosa for genitourinary reconstruction: A comparison of different collagen-based biodegradable membranes.

Tissue engineering is a method of growing importance regarding clinical application in the genitourinary region. One of the key factors in successfully development of an artificially tissue engineered mucosa equivalent (TEOM) is the optimal choice of the scaffold. Collagen scaffolds are regarded as gold standard in dermal tissue reconstruction. Four distinct collagen scaffolds were evaluated for the ability to support the development of an organotypical tissue architecture. TEOMs were established by seeding cocultures of primary oral epithelial cells and fibroblasts on four distinct collagen membranes. Cell viability was assessed by MTT-assay. The 3D architecture and functionality of the tissue engineered oral mucosa equivalents were evaluated by confocal laser-scanning microscopy and immunostaining. Cell viability was reduced on the TissuFoil E® membrane. A multi-stratified epithelial layer was established on all four materials, however the TEOMs on the Bio-Gide® scaffold showed the best fibroblast differentiation, secretion of tenascin and fibroblast migration into the membrane. The TEOMs generated on Bio-Gide® scaffold exhibited the optimal cellular organization into a cellular 3D network. Thus, the Bio-Gide® scaffold is a suitable matrix for engineering of mucosa substitutes in vitro.

Primary Mucosal Epithelial Cell Cultivation: A Reliable and Accelerated Isolation.

IMPACT STATEMENT: We illustrate a reliable and accelerated isolation routine for mucosal epithelial cells, which thereupon can be used for soft tissue engineering. This is highly important in the field of soft tissue engineering because mucosal equivalents are frequently usable in several surgical fields like gynecology, urology, otorhinolaryngology, ophthalmology, maxillofacial surgery, and many others. In this context the isolation of mucosal epithelial cells suitable for tissue engineering is mandatory. The reliable cultivation of mucosal or skin epithelial cells is challenging and there is currently no reproducible method. We demonstrate a solution for this problem by developing an accelerated and nevertheless reliable method.

Chronic social stress-induced hyperglycemia in mice couples individual stress susceptibility to impaired spatial memory.

Stringent glucose demands render the brain susceptible to disturbances in the supply of this main source of energy, and chronic stress may constitute such a disruption. However, whether stress-associated cognitive impairments may arise from disturbed glucose regulation remains unclear. Here we show that chronic social defeat (CSD) stress in adult male mice induces hyperglycemia and directly affects spatial memory performance. Stressed mice developed hyperglycemia and impaired glucose metabolism peripherally as well as in the brain (demonstrated by PET and induced metabolic bioluminescence imaging), which was accompanied by hippocampus-related spatial memory impairments. Importantly, the cognitive and metabolic phenotype pertained to a subset of stressed mice and could be linked to early hyperglycemia 2 days post-CSD. Based on this criterion, ∼40% of the stressed mice had a high-glucose (glucose >150 mg/dL), stress-susceptible phenotype. The relevance of this biomarker emerges from the effects of the glucose-lowering sodium glucose cotransporter 2 inhibitor empagliflozin, because upon dietary treatment, mice identified as having high glucose demonstrated restored spatial memory and normalized glucose metabolism. Conversely, reducing glucose levels by empagliflozin in mice that did not display stress-induced hyperglycemia (resilient mice) impaired their default-intact spatial memory performance. We conclude that hyperglycemia developing early after chronic stress threatens long-term glucose homeostasis and causes spatial memory dysfunction. Our findings may explain the comorbidity between stress-related and metabolic disorders, such as depression and diabetes, and suggest that cognitive impairments in both types of disorders could originate from excessive cerebral glucose accumulation.

Comparative metabolic analysis in head and neck cancer and the normal gingiva.

OBJECTIVES: Chronic accumulation of lactate in malignant tumor tissue is associated with increased malignancy and radioresistance. For this study, biopsies of primary head and neck squamous cell carcinoma (HNSCC) and of the normal gingiva of the same patient were compared via metabolic profiling to the healthy gingiva from cancer-free patients. MATERIALS AND METHODS: Cryobiopsies of 140 HNSCC patients were used to determine ATP, lactate, and glucose concentrations of the tumor and normal gingiva via induced metabolic bioluminescence imaging (imBI). Additionally, these metabolites were quantified in a collective of 79 healthy (non-tumor-bearing) patients. Furthermore, tumor samples were analyzed via immunofluorescence imaging and quantitative real-time PCR for the expression of lactate and glucose transporters. RESULTS: There were significant differences in ATP concentrations detectable between the tumor, normal gingiva of tumor patients, and gingiva from healthy patients. Lactate concentrations were significantly increased in tumor tissue compared to the normal gingiva of tumor patients as well as the gingiva from healthy patients. Concerning glucose, there was a significant decrease in glucose concentrations detectable in the tumor biopsies compared to the normal gingiva of tumor patients. On the other hand, tumor samples from patients revealed significantly elevated relative expression levels of monocarboxylate transporters (MCT-1 and MCT-4), as well as glucose transporters (GLUT-1 and GLUT-3) compared to the corresponding normal gingiva of each patient. CONCLUSIONS: We could demonstrate that the lactate concentration in HNSCC correlates with primary tumor (T) stage. CLINICAL RELEVANCE: The aim of this study was to identify metabolic parameters to improve early cancer diagnosis, allow predictions on the degree of malignancy, and contribute to a personalized tumor therapy.

Uncovering Metabolic Effects of Anti-angiogenic Therapy in Tumors by Induced Metabolic Bioluminescence Imaging.

Induced metabolic bioluminescence imaging (imBI) is an imaging technique which enables detection of various metabolites associated with glycolysis in tumor sections. Signals captured by imBI can be used to chart the topographic distribution of lactate, glucose, pyruvate, and ATP and quantify their absolute amount. ImBi can enable us to perform metabolic classification of tumors as well as to detect metabolic changes in the glycolytic pathway associated with certain therapies, such as anti-angiogenic drugs.

LDHA-Associated Lactic Acid Production Blunts Tumor Immunosurveillance by T and NK Cells.

Elevated lactate dehydrogenase A (LDHA) expression is associated with poor outcome in tumor patients. Here we show that LDHA-associated lactic acid accumulation in melanomas inhibits tumor surveillance by T and NK cells. In immunocompetent C57BL/6 mice, tumors with reduced lactic acid production (Ldhalow) developed significantly slower than control tumors and showed increased infiltration with IFN-γ-producing T and NK cells. However, in Rag2-/-γc-/- mice, lacking lymphocytes and NK cells, and in Ifng-/- mice, Ldhalow and control cells formed tumors at similar rates. Pathophysiological concentrations of lactic acid prevented upregulation of nuclear factor of activated T cells (NFAT) in T and NK cells, resulting in diminished IFN-γ production. Database analyses revealed negative correlations between LDHA expression and T cell activation markers in human melanoma patients. Our results demonstrate that lactic acid is a potent inhibitor of function and survival of T and NK cells leading to tumor immune escape.

Lactate-An Integrative Mirror of Cancer Metabolism.

The technique of induced metabolic bioluminescence imaging (imBI) has been developed to obtain a "snapshot" of the momentary metabolic status of biological tissues. Using cryosections of snap-frozen tissue specimens, imBI combines highly specific and sensitive in situ detection of metabolites with a spatial resolution on a microscopic level and with metabolic imaging in relation to tissue histology. Here, we present the application of imBI in human colorectal cancer. Comparing the metabolic information of one biopsy with that of 2 or 3 biopsies per individual cancer, the classification into high versus low lactate tumors, reflecting different glycolytic activities, based on a single biopsy was in agreement with the result from multiple biopsies in 83 % of all cases. We further demonstrate that the metabolic status of tumor tissue can be preserved at least over 10 years by storage in liquid nitrogen, but not by storage at -80 °C. This means that tissue banking with long-term preservation of the metabolic status is possible at -180 °C, which may be relevant for studies on long-term survival of cancer patients. As with other tumor entities, tissue lactate concentration was shown to be correlated with tumor development and progression in colorectal cancer. At first-time diagnosis, lactate values were low in rectal normal tissue and adenomas, were significantly elevated to intermediate levels in non-metastatic adenocarcinomas, and were very high in carcinomas with distant metastasis. There was an inverse behavior of tissue glucose concentration under corresponding conditions. The expression level of monocarboxylate transporter-4 (MCT4) was positively correlated with the tumor lactate concentration and may thus contribute to high lactate tumors being associated with a high degree of malignancy.

Feasibility of induced metabolic bioluminescence imaging in advanced ovarian cancer patients: first results of a pilot study.

PURPOSE: The precise determination of energy metabolites is challenged by the heterogeneity of their distribution, their rapid changes after surgical resection and the architectural complexity of malignancies. Induced metabolic bioluminescence imaging (imBI) allows to determine energy metabolites in tissue sections and to co-localize these with histological structures based on consecutive sections stained with HE. In this prospective pilot study patients with suspected advanced ovarian cancer (OC) were enrolled to prove the feasibility of imBI. METHODS: During surgery, suspicious peritoneal metastases were resected and transferred in liquid nitrogen within 30 s. ATP, glucose and lactate concentrations were measured. Furthermore, the expression of monocarboxylate transporters MCT1 and MCT4 was determined by immunofluorescence staining. RESULTS: 16 patients were screened, 12 entered the study. Final histological assessment revealed ten malignant and two benign peritoneal lesions. In all 12 cases high concentrations of ATP suggested that energy metabolism was not altered by the surgical and transport procedures (mean 0.56 µmol/g, range 0.24-1.21 µmol/g). The mean concentration of glucose was 1.95 µmol/g (range 0.58-4.71 µmol/g). The concentration of lactate was drastically higher in the ten OC cases (mean 24.79 µmol/g, range 17.51-37.16 µmol/g) compared to the benign samples (mean 5.98 µmol/g, range 5.43-6.54 µmol/g). Lactate concentrations seem to correlate with MCT1 (spearman rank correlation ρ = 0.624, 0.05 > p > 0.025), but not with MCT4 (spearman rank correlation ρ = 0.018, p > 0.1). CONCLUSIONS: ImBI is feasible in peritoneal metastases of OC and encourages further effort to elucidate the role of glucose, lactate, MCT1 and MCT4 in OC.

Differential Superiority of Heavy Charged-Particle Irradiation to X-Rays: Studies on Biological Effectiveness and Side Effect Mechanisms in Multicellular Tumor and Normal Tissue Models.

This review is focused on the radiobiology of carbon ions compared to X-rays using multicellular models of tumors and normal mucosa. The first part summarizes basic radiobiological effects, as observed in cancer cells. The second, more clinically oriented part of the review, deals with radiation-induced cell migration and mucositis. Multicellular spheroids from V79 hamster cells were irradiated with X-rays or carbon ions under ambient or restricted oxygen supply conditions. Reliable oxygen enhancement ratios could be derived to be 2.9, 2.8, and 1.4 for irradiation with photons, (12)C(+6) in the plateau region, and (12)C(+6) in the Bragg peak, respectively. Similarly, a relative biological effectiveness of 4.3 and 2.1 for ambient pO2 and hypoxia was obtained, respectively. The high effectiveness of carbon ions was reflected by an enhanced accumulation of cells in G2/M and a dose-dependent massive induction of apoptosis. These data clearly show that heavy charged particles are more efficient in sterilizing tumor cells than conventional irradiation even under hypoxic conditions. Clinically relevant doses (3 Gy) of X-rays induced an increase in migratory activity of U87 but not of LN229 or HCT116 tumor cells. Such an increase in cell motility following irradiation in situ could be the source of recurrence. In contrast, carbon ion treatment was associated with a dose-dependent decrease in migration with all cell lines and under all conditions investigated. The radiation-induced loss of cell motility was correlated, in most cases, with corresponding changes in ß1 integrin expression. The photon-induced increase in cell migration was paralleled by an elevated phosphorylation status of the epidermal growth factor receptor and AKT-ERK1/2 pathway. Such a hyperphosphorylation did not occur during (12)C(+6) irradiation under all conditions registered. Comparing the gene toxicity of X-rays with that of particles using the γH2AX technique in organotypic cultures of the oral mucosa, the superior effectiveness of heavy ions was confirmed by a twofold higher number of foci per nucleus. However, proinflammatory signs were similar for both treatment modalities, e.g., the activation of NFκB and the release of IL6 and IL8. The presence of peripheral blood mononuclear cell increased the radiation-induced release of the proinflammatory cytokines by factors of 2-3. Carbon ions are part of the cosmic radiation. Long-term exposure to such particles during extended space flights, as planned by international space agencies, may thus impose a medical and safety risk on the astronauts by a potential induction of mucositis. In summary, particle irradiation is superior to gamma-rays due to a higher radiobiological effectiveness, a reduced hypoxia-induced radioresistance, a multicellular radiosensitization, and the absence of a radiation-induced cell motility. However, the potential of inducing mucositis is similar for both radiation types.

Quantitative Imaging of D-2-Hydroxyglutarate in Selected Histological Tissue Areas by a Novel Bioluminescence Technique.

Patients with malignant gliomas have a poor prognosis with average survival of less than 1 year. Whereas in other tumor entities the characteristics of tumor metabolism are successfully used for therapeutic approaches, such developments are very rare in brain tumors, notably in gliomas. One metabolic feature characteristic of gliomas, in particular diffuse astrocytomas and oligodendroglial tumors, is the variable content of D-2-hydroxyglutarate (D2HG), a metabolite that was discovered first in this tumor entity. D2HG is generated in large amounts due to various "gain-of-function" mutations in the isocitrate dehydrogenases IDH1 and IDH2. Meanwhile, D2HG has been detected in several other tumor entities, including intrahepatic bile-duct cancer, chondrosarcoma, acute myeloid leukemia, and angioimmunoblastic T-cell lymphoma. D2HG is barely detectable in healthy tissue (<0.1 mM), but its concentration increases up to 35 mM in malignant tumor tissues. Consequently, the "oncometabolite" D2HG has gained increasing interest in the field of tumor metabolism. To facilitate its quantitative measurement without loss of spatial resolution at a microscopical level, we have developed a novel bioluminescence assay for determining D2HG in sections of snap-frozen tissue. The assay was verified independently by photometric tests and liquid chromatography/mass spectrometry. The novel technique allows the microscopically resolved determination of D2HG in a concentration range of 0-10 µmol/g tissue (wet weight). In combination with the already established bioluminescence imaging techniques for ATP, glucose, pyruvate, and lactate, the novel D2HG assay enables a comparative characterization of the metabolic profile of individual tumors in a further dimension.

Lactate as a predictive marker for tumor recurrence in patients with head and neck squamous cell carcinoma (HNSCC) post radiation: a prospective study over 15 years.

OBJECTIVES: Lactate as a key regulator of the glycolytic phenotype has been recently described in fueling tumor growth and metastatic spread in head and neck squamous cell carcinoma (HNSCC). However, in context of tumor recurrence following adjuvant radiation, the underlying mechanisms remain uncertain. We therefore investigate the role of lactate towards radioresistance in HNSCC in this prospective study for the first time in vivo. MATERIALS AND METHODS: Herein, we analyzed biopsies of primary squamous cell carcinoma after surgery and adjuvant irradiation in 17 patients. Tumor tissue levels of ATP, glucose, and lactate were detected using induced metabolic bioluminescence imaging (imBI) and correlated with clinical data within an observation period of up to 15 years. RESULTS: High amounts of lactate levels in tumors of HNSCC are significantly negatively correlated with overall patient survival. Moreover, high expression of lactate in a primary tumor site is significantly correlated with tumor recurrence post radiation, whereas ATP and/or glucose showed no such correlation. CONCLUSION: Lactate can be seen not only as a waste product of altered glycolytic metabolism but also as a key master of malignancy as well as resistance mechanism towards irradiation. CLINICAL RELEVANCE: High expression of lactate levels in tumor tissue, obtained by metabolic bioluminescence imaging, may therefore serve as a predictor for overall and recurrence-free survival and could represent a future biomarker in the validation of adjuvant irradiation.

VEGF-targeted therapy stably modulates the glycolytic phenotype of tumor cells.

Anti-VEGF therapy perturbs tumor metabolism, severely impairing oxygen, glucose, and ATP levels. In this study, we investigated the effects of anti-VEGF therapy in multiple experimental tumor models that differ in their glycolytic phenotypes to gain insights into optimal modulation of the metabolic features of this therapy. Prolonged treatments induced vascular regression and necrosis in tumor xenograft models, with highly glycolytic tumors becoming treatment resistant more rapidly than poorly glycolytic tumors. By PET imaging, prolonged treatments yielded an increase in both hypoxic and proliferative regions of tumors. A selection for highly glycolytic cells was noted and this metabolic shift was stable and associated with increased tumor aggressiveness and resistance to VEGF blockade in serially transplanted mice. Our results support the hypothesis that the highly glycolytic phenotype of tumor cells studied in xenograft models, either primary or secondary, is a cell-autonomous trait conferring resistance to VEGF blockade. The finding that metabolic traits of tumors can be selected by antiangiogenic therapy suggests insights into the evolutionary dynamics of tumor metabolism.

ERGO: a pilot study of ketogenic diet in recurrent glioblastoma.

Limiting dietary carbohydrates inhibits glioma growth in preclinical models. Therefore, the ERGO trial (NCT00575146) examined feasibility of a ketogenic diet in 20 patients with recurrent glioblastoma. Patients were put on a low-carbohydrate, ketogenic diet containing plant oils. Feasibility was the primary endpoint, secondary endpoints included the percentage of patients reaching urinary ketosis, progression-free survival (PFS) and overall survival. The effects of a ketogenic diet alone or in combination with bevacizumab was also explored in an orthotopic U87MG glioblastoma model in nude mice. Three patients (15%) discontinued the diet for poor tolerability. No serious adverse events attributed to the diet were observed. Urine ketosis was achieved at least once in 12 of 13 (92%) evaluable patients. One patient achieved a minor response and two patients had stable disease after 6 weeks. Median PFS of all patients was 5 (range, 3-13) weeks, median survival from enrollment was 32 weeks. The trial allowed to continue the diet beyond progression. Six of 7 (86%) patients treated with bevacizumab and diet experienced an objective response, and median PFS on bevacizumab was 20.1 (range, 12-124) weeks, for a PFS at 6 months of 43%. In the mouse glioma model, ketogenic diet alone had no effect on median survival, but increased that of bevacizumab-treated mice from 52 to 58 days (p<0.05). In conclusion, a ketogenic diet is feasible and safe but probably has no significant clinical activity when used as single agent in recurrent glioma. Further clinical trials are necessary to clarify whether calorie restriction or the combination with other therapeutic modalities, such as radiotherapy or anti-angiogenic treatments, could enhance the efficacy of the ketogenic diet.

MYCN and survivin cooperatively contribute to malignant transformation of fibroblasts.

The oncogenes MYCN and survivin (BIRC5) maintain aggressiveness of diverse cancers including sarcomas. To investigate whether these oncogenes cooperate in initial malignant transformation, we transduced them into Rat-1 fibroblasts. Indeed, survivin enhanced MYCN-driven contact-uninhibited and anchorage-independent growth in vitro. Importantly, upon subcutaneous transplantation into mice, cells overexpressing both instead of either one of the oncogenes generated tumors with shortened latency, marked anaplasia and an increased proliferation-to-apoptosis ratio resulting in accelerated growth. Mechanistically, the increased tumorigenicity was associated with an enhanced Warburg effect and a hypoxia inducible factor 1α linked vascular remodeling. This cooperation between MYCN and survivin may be important in the genesis of several cancers.

The Microenvironment of Cervical Carcinoma Xenografts: Associations with Lymph Node Metastasis and Its Assessment by DCE-MRI.

Poor disease-free and overall survival rates in locally advanced cervical cancer are associated with a tumor micro-environment characterized by extensive hypoxia, interstitial hypertension, and high lactate concentrations. The potential of gadolinium diethylenetriamine pentaacetic acid-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in assessing the microenvironment and microenvironment-associated aggressiveness of cervical carcinomas was investigated in this preclinical study. CK-160 and TS-415 cervical carcinoma xenografts were used as tumor models. DCE-MRI was carried out at 1.5 T, and parametric images of K (trans) and v e were produced by pharmacokinetic analysis of the DCE-MRI series. Pimonidazole was used as a marker of hypoxia. A Millar catheter was used to measure tumor interstitial fluid pressure (IFP). The concentrations of glucose, adenosine triphosphate (ATP), and lactate were measured by induced metabolic bioluminescence imaging. High incidence of lymph node metastases was associated with high hypoxic fraction and high lactate concentration in CK-160 tumors and with high IFP and high lactate concentration in TS-415 tumors. Low K (trans) was associated with high hypoxic fraction, low glucose concentration, and high lactate concentration in tumors of both lines and with high incidence of metastases in CK-160 tumors. Associations between v e and microenvironmental parameters or metastatic propensity were not detected in any of the tumor lines. Taken together, this preclinical study suggests that K (trans) is a potentially useful biomarker for poor outcome of treatment in advanced cervical carcinoma. The possibility that K (trans) may be used to identify patients with cervical cancer who are likely to benefit from particularly aggressive treatment merits thorough clinical investigations.

Synthetic lethal metabolic targeting of cellular senescence in cancer therapy.

Activated oncogenes and anticancer chemotherapy induce cellular senescence, a terminal growth arrest of viable cells characterized by S-phase entry-blocking histone 3 lysine 9 trimethylation (H3K9me3). Although therapy-induced senescence (TIS) improves long-term outcomes, potentially harmful properties of senescent tumour cells make their quantitative elimination a therapeutic priority. Here we use the Eµ-myc transgenic mouse lymphoma model in which TIS depends on the H3K9 histone methyltransferase Suv39h1 to show the mechanism and therapeutic exploitation of senescence-related metabolic reprogramming in vitro and in vivo. After senescence-inducing chemotherapy, TIS-competent lymphomas but not TIS-incompetent Suv39h1(-) lymphomas show increased glucose utilization and much higher ATP production. We demonstrate that this is linked to massive proteotoxic stress, which is a consequence of the senescence-associated secretory phenotype (SASP) described previously. SASP-producing TIS cells exhibited endoplasmic reticulum stress, an unfolded protein response (UPR), and increased ubiquitination, thereby targeting toxic proteins for autophagy in an acutely energy-consuming fashion. Accordingly, TIS lymphomas, unlike senescence models that lack a strong SASP response, were more sensitive to blocking glucose utilization or autophagy, which led to their selective elimination through caspase-12- and caspase-3-mediated endoplasmic-reticulum-related apoptosis. Consequently, pharmacological targeting of these metabolic demands on TIS induction in vivo prompted tumour regression and improved treatment outcomes further. These findings unveil the hypercatabolic nature of TIS that is therapeutically exploitable by synthetic lethal metabolic targeting.

Semiautomatic growth analysis of multicellular tumor spheroids.

Multicellular tumor spheroids (MCTS) are routinely employed as three-dimensional in vitro models to study tumor biology. Cultivation of MCTS in spinner flasks provides better growing conditions, especially with regard to the availability of nutrients and oxygen, when compared with microtiter plates. The main endpoint of drug response experiments is spheroid size. It is common practice to analyze spheroid size manually with a microscope and an ocular micrometer. This requires removal of some spheroids from the flask, which entails major limitations such as loss of MCTS and the risk of contamination. With this new approach, the authors present an efficient and highly reproducible method to analyze the size of complete MCTS populations in culture containers with transparent, flat bottoms. MCTS sediments are digitally scanned and spheroid volumes are calculated by computerized image analysis. The equipment includes regular office hardware (personal computer, flatbed scanner) and software (Adobe Photoshop, Microsoft Excel, ImageJ). The accuracy and precision of the method were tested using industrial precision steel beads with known diameter. In summary, in comparison with other methods, this approach provides benefits in terms of semiautomation, noninvasiveness, and low costs.

Differential utilization of ketone bodies by neurons and glioma cell lines: a rationale for ketogenic diet as experimental glioma therapy.

BACKGROUND: Even in the presence of oxygen, malignant cells often highly depend on glycolysis for energy generation, a phenomenon known as the Warburg effect. One strategy targeting this metabolic phenotype is glucose restriction by administration of a high-fat, low-carbohydrate (ketogenic) diet. Under these conditions, ketone bodies are generated serving as an important energy source at least for non-transformed cells. METHODS: To investigate whether a ketogenic diet might selectively impair energy metabolism in tumor cells, we characterized in vitro effects of the principle ketone body 3-hydroxybutyrate in rat hippocampal neurons and five glioma cell lines. In vivo, a non-calorie-restricted ketogenic diet was examined in an orthotopic xenograft glioma mouse model. RESULTS: The ketone body metabolizing enzymes 3-hydroxybutyrate dehydrogenase 1 and 2 (BDH1 and 2), 3-oxoacid-CoA transferase 1 (OXCT1) and acetyl-CoA acetyltransferase 1 (ACAT1) were expressed at the mRNA and protein level in all glioma cell lines. However, no activation of the hypoxia-inducible factor-1α (HIF-1α) pathway was observed in glioma cells, consistent with the absence of substantial 3-hydroxybutyrate metabolism and subsequent accumulation of succinate. Further, 3-hydroxybutyrate rescued hippocampal neurons from glucose withdrawal-induced cell death but did not protect glioma cell lines. In hypoxia, mRNA expression of OXCT1, ACAT1, BDH1 and 2 was downregulated. In vivo, the ketogenic diet led to a robust increase of blood 3-hydroxybutyrate, but did not alter blood glucose levels or improve survival. CONCLUSION: In summary, glioma cells are incapable of compensating for glucose restriction by metabolizing ketone bodies in vitro, suggesting a potential disadvantage of tumor cells compared to normal cells under a carbohydrate-restricted ketogenic diet. Further investigations are necessary to identify co-treatment modalities, e.g. glycolysis inhibitors or antiangiogenic agents that efficiently target non-oxidative pathways.

Inhibition of tumor lactate oxidation: consequences for the tumor microenvironment.

BACKGROUND AND PURPOSE: Tumor cells are recognized as being highly glycolytic. However, recently it was suggested that lactate produced in hypoxic tumor areas may be taken up by the monocarboxylate transporter MCT1 and oxidized in well-oxygenated tumor parts. Furthermore, it was shown that inhibition of lactate oxidation using the MCT1 inhibitor α-cyano-hydroxycinnamate (CHC) can radio-sensitize tumors possibly by forcing a switch from lactate oxidization to glycolysis in oxygenated cells, which in turn improves tumor oxygenation and indirectly kills radio-resistant hypoxic tumor cells from glucose starvation. MATERIAL AND METHODS: To provide direct evidence for the existence of a targetable energetic symbiosis, mice bearing SiHa or FaDu(dd) tumors were treated with CHC for different time periods. One hour prior to sacrifice, mice were administered with the glucose analog fluorodeoxyglucose (FDG) and the hypoxia-marker pimonidazole. Tumor cryosections were analyzed for regional glucose retention (FDG autoradiograms), hypoxia (pimonidazole retention) and glucose and lactate levels (bioluminescence imaging). RESULTS: Treatment did not influence metabolite concentrations, necrosis or extent of hypoxia, but pixel-by-pixel analysis comparing FDG retention and hypoxia (a measure of the apparent in vivo Pasteur effect) showed that CHC treatment caused a transient reduction in the Pasteur effect in FaDu(dd) 1.5 h following CHC administration whereas a reduction was only observed in SiHa following repeated treatments. CONCLUSIONS: In summary, our data show that CHC is able to influence the intratumoral distribution of glucose use between hypoxic and non-hypoxic tumor areas. That is in accordance with a functional tumor lactate-shuttle, but the absence of any detectable changes in hypoxic extent and tissue metabolites was unexpected and warrants further investigation.

Lactate enhances motility of tumor cells and inhibits monocyte migration and cytokine release.

In solid malignant tumors, lactate has been identified as a prognostic parameter for metastasis and overall survival of patients. To investigate the effects of lactate on tumor cell migration, Boyden chamber assays were applied. We could show here that lactate enhances tumor cell motility of head and neck carcinoma cell lines significantly in a dose-dependent manner. The changes in tumor cell migration could be attributed to L-lactate or a conversion of lactate to pyruvate, as only these two substances were able to increase migration. Addition of D-lactate or changes in osmolarity or intracellular pH did not alter the migratory potential of the cells investigated. Because lactate was shown earlier to impair the penetration of dendritic cells in a tumor spheroid model, which is contrary to the response of the malignant cell population in the present study, we included blood monocytes in our assay as a highly motile immune cell type and precursor of tumor-associated macrophages. Interestingly, high levels of L-lactate (20 mM) at a pH of 7.4 inhibited monocyte migration in the Boyden chamber system. In addition, cytokine release of TNF and IL-6 was inhibited. The obtained data suggest that high lactate content promotes tumor progression by contributing to the phenomenon of tumor immune escape and by enhancing the migratory potential of the malignant cell population which may directly be coupled to a higher incidence of metastasis.